In 2013, the CDC designated carbapenem-resistant Enterobacteriaceae (CRE) an Urgent Threat, and in 2017, the WHO designated it a Priority 1 ?critical superbug?. As few therapies remain to treat CRE, the risk of ?pan- resistant? CRE, untreatable by any currently available antibiotic, increases. Entirely new agents with novel mechanisms of action (MOA) not cross-resistant to SOC agents languish. Our proposal aims to develop an O- antigen biosynthetic inhibitory agent that potentiates serum-mediated killing (SMK) and is efficacious in a CRE rodent model of infection. We have shown that the O-antigen biosynthesis gene wecA, a nonessential gene under standard growth conditions, is essential for growth and pathogenesis in the presence of mammalian serum. Our proposal outlines a plan to develop synthetic inhibitors of WecA that we previously discovered and optimized to inhibit the Gram-positive WecA ortholog, TarO. Our Aims are: Aim 1 (Phase 1; Ph1). Screening, MOA studies, and Assay Development. (1) Complete screening of the tarocin library for SMK against E. coli (Ec), (2) confirm tarocins inhibit EcWecA as their MOA for eliciting SMK, (3) establish key target engagement (TE) screening assays, and (4) demonstrate that tarocin-induced SMK extends to K. pneumoniae (Kp). Milestone 1. Screen ~600 additional tarocin analogs for SMK activity, construct TE assays to support Ph2 optimization, and identify up to 4 distinct tarocin subseries demonstrating i) > 4-fold EC50 shift in SMK by EcWecA overexpression, ii) whole-cell dose-dependent depletion of O-a, iii) causal drugR mutations mapping to EcwecA, and iv) SMK activity against Kp DtolC to advance. Aim 2 (Ph2). Lead Identification/Optimization. Identify WecA-specific tarocins with potent WT Ec and Kp SMK by (1) empirically testing analogs for SMK against a panel of Ec and Kp permeability/efflux deficient mutants, (2) employing recent physicochemical rules of GN entry, and (3) exploring siderophore conjugation to drive SAR efforts. We will also optimize PK and drug-like properties. Milestone 2. Identify up to 3 top analogs demonstrating i) Ec/Kp WT activity (MIC in serum [MICs] < 1 ug/ml), ii) PK exposure to cover MICs for 4 hrs, iii) Ec TE and PE selectivity, iv) tarocinR mutation that maps to wecA in Kp to establish conserved MOA across bacterial species v) Ec/Kp FOR < 1 x 10-9 at 8X MICs, vi) MICs90 < 2 ug/ml (n=100 clinical isolates), and vii) > 90% HepG2 and HEK293 cell viability at 10X MICs, clean vs CYP/ion channels (>10 uM), and PanLabs IC50 > 10 uM. Aim 3 (Ph2). In vivo efficacy demonstration. (1) Optimize synthetic routes to efficiently prepare 3 analogs for formulation and dose-ranging rat PK studies, (2) identify formulation verhicles for the 3 analogs to enable oral and IV PK dosing in in vivo studies, and (3) demonstrate in vivo efficacy for our top compound in a rodent septicemia model using Ec and Kp strains. Milestone 3. Synthesize 500 mg (>95% purity) of up to 3 optimal analogs that best satisfy Aim 2 milestones, identify formulation in a safety approved vehicle that achieves 10X MICS90 target exposure, and demonstrate > 3 log reduction of Ec and Kp bacterial burden after 24h treatment.